Considering the state of the art, there are different studies which evidence the existence of antibodies that recognize lipids. For example, they have been detected in the serum of patients with antiphospholipid syndrome, as was described by Asherson et al. in their book “The antiphospholipid syndrome” in 1996 (CRC Press, Boca Raton). In the same way, antiphospholipid antibodies have been obtained from animals that were experimentally treated with lipids by active immunization, in accordance with Alving in 1992 (Biochim. Biophys. Acta 1113:307-322) and in animals that received antiphospholipid antibodies by passive immunization, as Tincani and Shoenfeld described in 1996 in the above mentioned book.
Anti-lipid antibodies have been classified into two major subgroups based on the method used for their determination. These groups are anti-cardiolipin antibodies and anticoagulant antibodies (Guglielmone y Fernandez, 1998, J. Rheumatol. 26:86-90).
Anti-cardiolipin antibodies are determined by methods using cardiolipin immobilized in a solid phase. Harris et al. in 1985 (Clin. Rheum. Dis. 11:591-609) described enzyme-linked immunosorbent assays and radioimmunoassays, better known by their respective abbreviations as ELISA and RIA. These have been broadly used in the above mentioned technique.
Anticoagulant antibodies are detected by measuring the prolongation in the coagulation time of plasma samples in vitro, according with Bevers et al. 1991 (Thromb. Haemost. 66:629-632). Some of these methods are: activated partial thromboplastin time (APTT), dilute Russell's viper venom time (dRVVT), protein C, and protein S, among others. In these methods, anticoagulant antibodies are bound to phosphatidylethanolamine or to phosphatidylserine, which are intermediary factors in the blood coagulation cascade, and when the concentration of these lipids decrease due to the immune reaction, the coagulation time is prolongated.
Anti-cardiolipin antibodies have the disadvantage of producing cross-reactions with other anionic lipids such as phosphatidylserine and phosphatidylglycerol. Due to their lack of specificity for a certain type of lipid, the above mentioned antibodies are generally known as antiphospholipid antibodies.
In addition, antibodies against phosphatidylethanolamine have been detected in the sera of patients with antiphospholipid syndrome. Also, antibodies against phosphatidylcholine are detected in patients with hemolytic anemia, as was described by Sugi and McIntyre (Blood 86:3083-3089) and Arvieux et al. (Thromb. Haemost. 74:1120-1125), respectively, in 1995.
On the other hand, some studies have demonstrated that the binding of antiphospholipid antibodies to the lipidic antigen increases in the presence of a plasma protein. For example, in 1990, McNeil et al., determined that the binding of antibodies to cardiolipin was markedly enhanced by the plasma protein b2-glycoprotein I or apoprotein H (Proc. Nat. Acad. Sci. USA 87:4120-4124). Additionally, some anti-cardiolipin antibodies are bound directly to b2-glycoprotein I, as was described by Roubey et al. in 1995 (J. Immunol. 154:954-960). These findings suggest that the anti-cardiolipin antibodies may recognize either a cryptic epitope on b2-glycoprotein I exposed on the complex of b2-glycoprotein I-cardiolipin, or b2-glycoprotein I alone but with a very low affinity towards the glycoprotein, as was described by Pengo et al. (1995, Thromb. Haemost. 73:29-34).
In accordance with these studies, it may be concluded that the binding of antiphospholipid antibodies to lipidic antigens is also associated with proteins. Sugi and McIntyre (op. cit., 1995) found that proteins called kininogens are involved in the binding of antibodies to phosphatidylethanolamine, whereas proteins that are bound to phosphatidylserine, such as prothrombin, protein C, protein S and annexin V, have been implicated in the binding of anticoagulant antibodies to phosphatidylserine, according to the studies in 1994 by Nakamura et al. (Biochim. Biophys. Res. Commun. 205:1488-1493) and by Roubey (Blood 84:2854-2867).
These studies indicate that the antigen of some antiphospholipid antibodies is really a complex formed by phospholipids and specific plasma proteins, but these proteins differ from those required for reactivity of antiphospholipid antibodies with cardiolipin. Nevertheless, in other studies, antiphospholipid antibodies that bind directly to the phospholipid have been identified, for example anti-cardiolipin antibodies that do not require b2-glycoprotein I. Such studies were carried out by McNeil et al. in 1989 (Br. J. Haematol. 73:506-513) and by Pengo and Basiolo in 1993 (Thromb. Res. 72:423-430).
On the other hand, some anti-cardiolipin antibodies, purified by affinity chromatography, do not show anticoagulant activity (McNeil et al., op. cit., 1989; Shi et al., 1993, Blood 81:1255-1262). However, other studies demonstrated that anti-cardiolipin and anticoagulant antibodies were removed by adsorption with cardiolipin (Pengo and Biasiolo, op. cit., 1993; Pierangeli et al. 1993, Br. J. Haematol. 85:124-132).
Additionally, studies in experimental animals, treated by passive or active immunization, employed methods for the detection of antiphospholipid antibodies which are the same as those described for the detection of human antiphospholipid antibodies. Furthermore, in these animal models, the different organs and tissues were analyzed by anatomical and histopathological studies, by immunofluorescent studies, and even by fetal resorption analysis in which the produced lesions in fetuses and placentas of the female animal models were also analyzed. These studies were performed by Tincani and Shoenfeld (op. cit. 1996) and by Shoenfeld and Ziporen (Lupus 7:S158-S161, 1998).
The previously mentioned studies show that the antiphospholipid antibodies described in human patients and in animal models have a broad specificity toward lipidic antigens. This broad specificity of the antibodies may be attributed, among other causes, to the lack of specificity of the methods used for the detection of the above described antibodies.
Such methods do not consider the chemical structure and the molecular association of lipidic antigens, as well as the chemical properties that the lipidic antigens have in nature. As a consequence, those methods use lipidic antigens where phospholipids are bound to artificial solid supports, such as in the ELISA and RIA methods, or they are in a molecular association that is not completely characterized, like in tests where the prolongation in coagulation time is detected.
There are only a few studies in which the molecular structure of the phospholipid employed as antigen has been considered, for example, the reports of Rauch et al. in 1989 and in 1998 (Thromb. Haemost. 62:892-896 and Thromb. Haemost. 80:936-941, respectively) and of Berard et al. (J. Lab. Clin. Med., 1993, 122:601-605). In these reports, the authors demonstrated that the anticoagulant activity of sera from some patients with systemic lupus erythematosus is inhibited by phosphatidylethanolamine which is associated in a hexagonal tubular II phase. This inhibition was not observed when the phospholipid was in a bilayer phase. However, the properties of the cellular membrane can not be related to the tubular association of phospholipids because this tubular lipidic association is practically incompatible with the vesicular structure of the cellular membrane, as different authors have established. In other words, in the lipidic antigens used in these studies, the phospholipids are in molecular arrangements that do not correspond to the molecular arrangements present in the cellular membrane.
Additionally, it is well known that the molecular structure of the plasma membrane of mammalian cells is an associated heteropolymer formed by phospholipids, glycolipids, cholesterol, proteins and glycoproteins, where the lipids are mainly in a bilayer molecular arrangement. Nevertheless, it is also known that lipids may have molecular arrangements different from a bilayer and that such arrangements depend on the molecular geometry of the lipids and the surrounding conditions.
Cylindrical shaped lipids, such as phosphatidate, phosphatidylglycerol, phosphatidylinositol, phosphatidyl-choline, phosphatidylserine, cardiolipin, sphingomyelin and diglucosyldiacylglycerides, are associated in closed bilayers, or liposomes in aqueous media. Cylindrical lipids constitute from 60 to 70% of membrane lipids.
On the other hand, the conic shaped lipids such as phosphatidylethanolamine, monoglucosyldiacylglycerides, and diacylglycerols, as well as the above mentioned lipids phosphatidate, cardiolipin, phosphatidylserine, and phosphatidylglycerol when in the presence of divalent cations, are assembled in the molecular phase known as hexagonal II (HII), which corresponds to tubular cylinders packed hexagonally. Inverted cone shaped lipids, such as lysophospholipids and gangliosides, are associated in micelles. Conic and inverted conic shaped lipids represent from 30 to 40% of membrane lipids.
Lipidic arrangements in hexagonal II or micellar phases, as well as any other structural arrangement of lipids that does not form a bilayer but is immersed in a bilayer, are, for the purposes of this invention, lipidic structures different from the lipidic bilayer, or lipidic particles, independent of the kind of lipids that are forming these structures.
In the same way, it is known that in the presence of divalent cations, drugs like chlorpromazine and procainamide, non-polar peptides, proteins such as the protein of the bacteriophage M13, cholesterol, lanthanum ions, as well as with changes in temperature and pH, the conic lipids form molecular arrangements different from a lipidic bilayer. These lipidic arrangements are of transient nature because when the concentration of the compounds that induced their formation diminishes or when the temperature or the pH changes again, the conic shaped lipids return to the bilayer arrangement as was described by Cullis et al., in 1991 (Membrane Fusion. Marcel Dekker, New York), by Baeza et al. in 1995 (Biochem. Cell Biol. 73:289-297) and Aguilar et al., in 1999 (J. Biol. Chem. 274:25193-25196). Lipidic bilayer molecular arrangements are observed as a smooth surface by cryofracture analysis.
Lipids in general are molecules with low immunogenicity, and of the two molecular arrangements that lipids may adopt in cellular membranes, it is considered that the lipidic bilayer is less immunogenic because it is the arrangement that mainly constitutes the matrix of all cellular membranes.
However, it is known that lipidic structures different from the bilayer, which are stabilized by divalent cations and are observed as protuberances on the smooth surface of the bilayer by cryofracture analysis, induce the formation of antibodies that recognize lipids that are associated in lipidic particles and do not react with lipids associated in a bilayer.
In connection with the above-mentioned studies, Baeza and their collaborators in 1995 (op. cit.) reported the production of liposomes having lipid molecular arrangements different from a bilayer, as well as the antigenic activity of these molecular arrangements, because they were able to obtain polyclonal antibodies to them. By means of cytofluorometric analysis of the immune reaction they were also able to identify the presence of lipidic structures in the liposomes described, using them to obtain anti-lipidic particles polyclonal antibodies in mice sera.
To do this, mice were immunized by introducing artificially formed lipidic particles, which when present in excess, caused the wanted immune reaction. Until now, it was believed that molecular arrangements different from a bilayer, lipidic particles, would be also poorly immunogenic when they are present in nature, for example in human and animal cells, because lipidic particles are transient and therefore would not be detected by immune systems.
Additionally, from analysis of the above mentioned studies, one can observe that cardiolipin is the only lipid that has been able to react with antibodies present in patients with antiphospholipid syndrome or associated illnesses, and that the other phospholipids usually present in the cellular membrane generally must be associated with proteins to react with the antibodies from these patients, or must be associated in a molecular arrangement incompatible with the molecular structure of the cellular membrane; with the exception of the studies of Baeza and their collaborators (op. cit. 1995) on anti-lipidic particle antibodies which react with a lipidic molecular arrangement similar to the one that has been described in cellular membranes.
In this respect, the presence in the sera of patients with antiphospholipid syndrome anti-cardiolipin antibodies, a mitochondrial lipid, anti-nuclear antibodies and anti-DNA antibodies, indicates the existence of previous events that caused immunologic damage to cellular membranes, with the disruption of the cells and presentation of intracellular components to the immunologic system, causing the corresponding immunologic reaction that contributes to the development of the syndrome. However, up to now there have not been studies which allow one to determine the events that cause the disruption of the cellular membrane. In other words, with the existent knowledge so far, it is impossible to detect anti-cardiolipin antibodies, anti-nuclear or even anti-DNA antibodies before damage has been caused to the cell, impeding an early diagnosis and treatment of the illnesses associated with the syndrome.
Additionally, in the Doctoral Thesis presented by Leopoldo Aguilar Dec. 17, 1997 (Determination of non-bilayer lipidic arrangements in liposomes and cellular membranes with monoclonal antibodies”, Doctoral Thesis, National School of Biological Sciences, National Polytechnic Institute, México) 5 sera from patients with primary antiphospholipid syndrome and 5 sera from patients with systemic lupus erythematosus were analyzed. The illnesses were corroborated by clinical characteristics that the patients presented and by means of the detection of anti-cardiolipin antibodies and of anti-nuclear antibodies, the latter in the case of the sick persons with lupus. The analyzed sera from all patients also contained anti-lipidic particles antibodies, detected according to the techniques of liposomal-ELISA and of liposomal cytofluorometry as described in the above mentioned Thesis.
This discovery, however, does not show any advantage for the early detection of the illnesses, since the presence of the antiphospholipid antibodies and of the anti-lipidic particles antibodies in those patients can be explained according with two hypothesis.
The first hypothesis assumes that an unknown factor causes the destruction of the cellular membrane, which promotes the formation of lipidic particles from membrane lipids that enter in contact with the immunologic system together with the intracellular components, consequently simultaneously forming anti-lipidic particle antibodies and anti-cardiolipin and anti-nuclear antibodies.
The second hypothesis assumes that lipidic particles are formed in the cellular membrane before its destruction, and the anti-lipidic particles antibodies formed destroy the membrane, exposing the intracellular components to the immunologic system and causing later formation of anti-cardiolipin and anti-nuclear antibodies.
This second hypothesis was proposed in the Master Thesis presented by Monica Lara on Aug. 20, 1999 (“Detection of anti-lipidic particles antibodies in patients with the anti-phospholipid syndrome,” Master Thesis, Escuela Nacional de Ciencias Biologicas [National School of Biological Sciences], Instituto Politécnico Nacional [National Polytechnic Institute], Mexico.
So far, neither of the two hypothesis has been demonstrated, which is of supreme importance for the treatment of the illnesses, since should the second hypothesis be proved, it would be possible to detect the above-mentioned illnesses in their early stages, and also prevention, cure or patient improvement from such illnesses would be possible.
Based on the above-mentioned hypothesis, it has been aimed to avoid the inconveniences of methods using induction and detection of antiphospholipid antibodies caused by the structure and molecular association of the antigens by using lipidic antigens with a structure and molecular association similar to that found in patients with illnesses associated with antiphospholipids antibodies. These novel lipidic antigens have been used for the induction and detection of anti-lipidic particle antibodies that allow an early diagnosis of these illnesses, as well as for the determination of physiologic states of the cell such as apoptosis (programmed cellular death) (Pittoni and Isenberg, 1998, Semin. Arthritis. Rheum. 28:163-178) and those associated with the cellular cycle (Go, G1, G2 and M) among others.